摘要：

通过对外泌体及其miRNA和乳腺相关泌乳因子的研究及乳汁中外泌体miRNA(bta-miR-146a、bta-miR-224)的差异表达,探索牛乳腺上皮细胞来源外泌体miRNA在亚临床型乳房炎早期诊断中的可行性,寻找亚临床型乳房炎早期诊断和治疗的新靶标,为奶牛乳房炎的早期防治提供理论依据和实践指导。运用20 μg/mL LPS刺激奶牛乳腺上皮细胞,体外构建亚临床型奶牛乳房炎细胞模型,检测体外模型中乳腺上皮细胞来源外泌体bta-miR-146a和bta-miR-224及泌乳基因的差异表达,进一步采集健康和患病奶牛乳汁并其检测外泌体中bta-miR-146a和bta-miR-224的差异表达。结果显示,20 μg/mL的LPS作用24 h即可成功建立亚临床型奶牛乳房炎细胞模型。在该模型中,产奶量和乳汁质量下降的重要标志因子ACSL1的表达量极显著下降。从乳腺上皮细胞培养液和乳汁中成功提取并鉴定了外泌体,运用透射电子显微镜也可以清晰地观察到外泌体的脂质双层膜结构,囊泡大小为30~150 nm。20 μg/mL LPS刺激乳腺上皮细胞和乳汁来源外泌体中bta-miR-146a和bta-miR-224的表达均显著降低。总之,牛奶来源外泌体bta-miR-146a和bta-miR-224可以作为奶牛乳房炎早期诊断的候选因子。

关键词: 奶牛, 乳房炎, 外泌体, bta-miR-146a, bta-miR-224

Abstract:

To explore the feasibility of bovine mammary epithelial cell-derived exocrine miRNAs in the early diagnosis of subclinical mastitis by studying exosomes and their miRNAs and mammary gland related lactation factors and the differential expression of exosomes miRNAs(bta-miR-146a,bta-miR-224)in milk,and to find new targets for the early diagnosis and treatment of subclinical mastitis,providing theoretical basis and practical guidance for the early prevention and treatment of cow mastitis.Therefore,20 μg/mL LPS was used to stimulate bovine mammary epithelial cells and construct an in vitro subclinical bovine mastitis cell model.Then,the differential expression of bta-miR-146a and bta-miR-224,along with milk genes in the exosomes derived from mammary epithelial cells were detected.Further,milk from healthy and diseased cows were collected to detect the differential expression of bta-miR-146a and bta-miR-224 in exosomes.The results showed that subclinical bovine mastitis cell models could be successfully established with 20 μg/mL LPS treatment for 24 h.In this model,the expression of ACSL1,an essential marker for decreased milk yield and quality,was significantly reduced.Exosomes derived from mammary epithelial cell culture media and milk were successfully extracted and identified,with the lipid bilayer structure of exosomes clearly visible under transmission electron microscopy,and vesicle sizes ranged between 30 and 150 nm.The expressions of bta-miR-146a and bta-miR-224 in exosomes derived from both mammary epithelial cells and milk were significantly reduced following 20 μg/mL LPS stimulation.In conclusion,bta-miR-146a and bta-miR-224 in milk-derived exosomes could serve as candidate factors for early diagnosis of bovine mastitis.

Key words: Cow, Mastitis, Exosome, bta-miR-146a, bta-miR-224